{"title":"新硫杆菌亚硫酸盐细胞色素c氧化还原酶的纯化及性质研究","authors":"A. Michael Charles , Isamu Suzuki","doi":"10.1016/0926-6593(66)90013-0","DOIUrl":null,"url":null,"abstract":"<div><p></p><ul><li><span>1.</span><span><p>1. Sulfite oxidase (sulfite: cytochrome <span><math><mtext>c</mtext></math></span> oxidoreductase) was purified from <em>Thiobacillus novellus</em> and the properties were studied.</p></span></li><li><span>2.</span><span><p>2. Sulfite oxidase did not require AMP for sulfite oxidation and was distinct from adenosine phosphosulfate reductase of <em>Thiobacillus thioparus</em>.</p></span></li><li><span>3.</span><span><p>3. The enzyme reduced either ferricyanide or cytochrome <span><math><mtext>c</mtext></math></span> with sulfite stoichiometrically, reducing 2 moles of the electron acceptor for every mole of sulfite.</p></span></li><li><span>4.</span><span><p>4. the pH optimum of enzyme was around 8 with potassium phosphate buffers.</p></span></li><li><span>5.</span><span><p>5. No cofactor requirements were demonstrated for the enzyme activity.</p></span></li><li><span>6.</span><span><p>6. The enzyme was specific for sulfite as substrate. Thiosulfate, cysteine, GSH, NANO<sub>2</sub> and NH<sub>2</sub>OH did not replace sulfite.</p></span></li><li><span>7.</span><span><p>7. The <span><math><mtext>K</mtext><msub><mi></mi><mn>m</mn></msub></math></span> for sulfite at pH 8.0 was determined as 4·10<sup>−5</sup> M and 2·10<sup>−5</sup> M with cytochrome <span><math><mtext>c</mtext></math></span> and ferricyanide as electron acceptors, respectively. At pH 6.5 the <span><math><mtext>K</mtext><msub><mi></mi><mn>m</mn></msub></math></span> for sulfite was 2·10<sup>−6</sup> M with cytochrome <span><math><mtext>c</mtext></math></span>.</p></span></li><li><span>8.</span><span><p>8. Various salts and buffers inhibited the enzyme activity. With NaCl the inhibition was found to be competitive with respect to sulfite. The <span><math><mtext>K</mtext><msub><mi></mi><mn>i</mn></msub></math></span> was calculated as 4.5· 10<sup>−3</sup> M.</p></span></li><li><span>9.</span><span><p>9. The enzymes was strongly inhibited by various sulfhydryl inhibitors inlcuding <span><math><mtext>p-</mtext><mtext>hydroxymercuribenzoate</mtext></math></span> and <span><math><mtext>N-</mtext><mtext>ethylmaleimide</mtext></math></span>. The inhibition by <span><math><mtext>p-</mtext><mtext>hydroxymercuribenzoate</mtext></math></span> was completely reversed by GSH.</p></span></li><li><span>10.</span><span><p>10. Methylene blue, NAD<sup>+</sup>, NADP<sup>+</sup> and O<sub>2</sub> did not replace Fe(CN)<sub>6</sub><sup>3−</sup> or cytochrome <span><math><mtext>c</mtext></math></span> as electron acceptor for sulfite oxidase. <em>T. novellus</em> cytochrome <span><math><mtext>c</mtext></math></span> was reduced by the enzyme and sulfite and subsequently oxidized by <em>T. novellus</em> cytochrome oxidase.</p></span></li><li><span>11.</span><span><p>11. Oxidative phosphorylation coupled to sulfite oxidation with a low P/O ratio was demonstrated in cell-free extracts of <em>T. novellus</em>.</p></span></li><li><span>12.</span><span><p>12. It is concluded that sulfite oxidase of <em>T. novellus</em> catalyzes the oxidation of sulfite according to the following equation. <span><math><mtext>SO</mtext><msub><mi></mi><mn>3</mn></msub><msup><mi></mi><mn>2−</mn></msup><mtext> + 2 </mtext><mtext>cyt</mtext><mtext> c </mtext><mtext>Fe</mtext><msup><mi></mi><mn>3+</mn></msup><mtext> + </mtext><mtext>H</mtext><msub><mi></mi><mn>2</mn></msub><mtext>O</mtext><mtext> → </mtext><mtext>SO</mtext><msub><mi></mi><mn>2</mn></msub><msup><mi></mi><mn>2−</mn></msup><mtext> + 2 </mtext><mtext>cyt</mtext><mtext> c </mtext><mtext>Fe</mtext><msup><mi></mi><mn>2−</mn></msup><mtext> + 2 </mtext><mtext>H</mtext><msup><mi></mi><mn>+</mn></msup></math></span></p></span></li></ul></div>","PeriodicalId":100160,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Enzymology and Biological Oxidation","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"1966-12-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0926-6593(66)90013-0","citationCount":"46","resultStr":"{\"title\":\"Purification and properties of sulfite: Cytochrome c oxido-reductase from Thiobacillus novellus\",\"authors\":\"A. Michael Charles , Isamu Suzuki\",\"doi\":\"10.1016/0926-6593(66)90013-0\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p></p><ul><li><span>1.</span><span><p>1. Sulfite oxidase (sulfite: cytochrome <span><math><mtext>c</mtext></math></span> oxidoreductase) was purified from <em>Thiobacillus novellus</em> and the properties were studied.</p></span></li><li><span>2.</span><span><p>2. Sulfite oxidase did not require AMP for sulfite oxidation and was distinct from adenosine phosphosulfate reductase of <em>Thiobacillus thioparus</em>.</p></span></li><li><span>3.</span><span><p>3. The enzyme reduced either ferricyanide or cytochrome <span><math><mtext>c</mtext></math></span> with sulfite stoichiometrically, reducing 2 moles of the electron acceptor for every mole of sulfite.</p></span></li><li><span>4.</span><span><p>4. the pH optimum of enzyme was around 8 with potassium phosphate buffers.</p></span></li><li><span>5.</span><span><p>5. No cofactor requirements were demonstrated for the enzyme activity.</p></span></li><li><span>6.</span><span><p>6. The enzyme was specific for sulfite as substrate. Thiosulfate, cysteine, GSH, NANO<sub>2</sub> and NH<sub>2</sub>OH did not replace sulfite.</p></span></li><li><span>7.</span><span><p>7. The <span><math><mtext>K</mtext><msub><mi></mi><mn>m</mn></msub></math></span> for sulfite at pH 8.0 was determined as 4·10<sup>−5</sup> M and 2·10<sup>−5</sup> M with cytochrome <span><math><mtext>c</mtext></math></span> and ferricyanide as electron acceptors, respectively. At pH 6.5 the <span><math><mtext>K</mtext><msub><mi></mi><mn>m</mn></msub></math></span> for sulfite was 2·10<sup>−6</sup> M with cytochrome <span><math><mtext>c</mtext></math></span>.</p></span></li><li><span>8.</span><span><p>8. Various salts and buffers inhibited the enzyme activity. With NaCl the inhibition was found to be competitive with respect to sulfite. The <span><math><mtext>K</mtext><msub><mi></mi><mn>i</mn></msub></math></span> was calculated as 4.5· 10<sup>−3</sup> M.</p></span></li><li><span>9.</span><span><p>9. The enzymes was strongly inhibited by various sulfhydryl inhibitors inlcuding <span><math><mtext>p-</mtext><mtext>hydroxymercuribenzoate</mtext></math></span> and <span><math><mtext>N-</mtext><mtext>ethylmaleimide</mtext></math></span>. The inhibition by <span><math><mtext>p-</mtext><mtext>hydroxymercuribenzoate</mtext></math></span> was completely reversed by GSH.</p></span></li><li><span>10.</span><span><p>10. Methylene blue, NAD<sup>+</sup>, NADP<sup>+</sup> and O<sub>2</sub> did not replace Fe(CN)<sub>6</sub><sup>3−</sup> or cytochrome <span><math><mtext>c</mtext></math></span> as electron acceptor for sulfite oxidase. <em>T. novellus</em> cytochrome <span><math><mtext>c</mtext></math></span> was reduced by the enzyme and sulfite and subsequently oxidized by <em>T. novellus</em> cytochrome oxidase.</p></span></li><li><span>11.</span><span><p>11. Oxidative phosphorylation coupled to sulfite oxidation with a low P/O ratio was demonstrated in cell-free extracts of <em>T. novellus</em>.</p></span></li><li><span>12.</span><span><p>12. It is concluded that sulfite oxidase of <em>T. novellus</em> catalyzes the oxidation of sulfite according to the following equation. <span><math><mtext>SO</mtext><msub><mi></mi><mn>3</mn></msub><msup><mi></mi><mn>2−</mn></msup><mtext> + 2 </mtext><mtext>cyt</mtext><mtext> c </mtext><mtext>Fe</mtext><msup><mi></mi><mn>3+</mn></msup><mtext> + </mtext><mtext>H</mtext><msub><mi></mi><mn>2</mn></msub><mtext>O</mtext><mtext> → </mtext><mtext>SO</mtext><msub><mi></mi><mn>2</mn></msub><msup><mi></mi><mn>2−</mn></msup><mtext> + 2 </mtext><mtext>cyt</mtext><mtext> c </mtext><mtext>Fe</mtext><msup><mi></mi><mn>2−</mn></msup><mtext> + 2 </mtext><mtext>H</mtext><msup><mi></mi><mn>+</mn></msup></math></span></p></span></li></ul></div>\",\"PeriodicalId\":100160,\"journal\":{\"name\":\"Biochimica et Biophysica Acta (BBA) - Enzymology and Biological Oxidation\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"1966-12-14\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1016/0926-6593(66)90013-0\",\"citationCount\":\"46\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biochimica et Biophysica Acta (BBA) - Enzymology and Biological Oxidation\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/0926659366900130\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochimica et Biophysica Acta (BBA) - Enzymology and Biological Oxidation","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/0926659366900130","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Purification and properties of sulfite: Cytochrome c oxido-reductase from Thiobacillus novellus
1.
1. Sulfite oxidase (sulfite: cytochrome oxidoreductase) was purified from Thiobacillus novellus and the properties were studied.
2.
2. Sulfite oxidase did not require AMP for sulfite oxidation and was distinct from adenosine phosphosulfate reductase of Thiobacillus thioparus.
3.
3. The enzyme reduced either ferricyanide or cytochrome with sulfite stoichiometrically, reducing 2 moles of the electron acceptor for every mole of sulfite.
4.
4. the pH optimum of enzyme was around 8 with potassium phosphate buffers.
5.
5. No cofactor requirements were demonstrated for the enzyme activity.
6.
6. The enzyme was specific for sulfite as substrate. Thiosulfate, cysteine, GSH, NANO2 and NH2OH did not replace sulfite.
7.
7. The for sulfite at pH 8.0 was determined as 4·10−5 M and 2·10−5 M with cytochrome and ferricyanide as electron acceptors, respectively. At pH 6.5 the for sulfite was 2·10−6 M with cytochrome .
8.
8. Various salts and buffers inhibited the enzyme activity. With NaCl the inhibition was found to be competitive with respect to sulfite. The was calculated as 4.5· 10−3 M.
9.
9. The enzymes was strongly inhibited by various sulfhydryl inhibitors inlcuding and . The inhibition by was completely reversed by GSH.
10.
10. Methylene blue, NAD+, NADP+ and O2 did not replace Fe(CN)63− or cytochrome as electron acceptor for sulfite oxidase. T. novellus cytochrome was reduced by the enzyme and sulfite and subsequently oxidized by T. novellus cytochrome oxidase.
11.
11. Oxidative phosphorylation coupled to sulfite oxidation with a low P/O ratio was demonstrated in cell-free extracts of T. novellus.
12.
12. It is concluded that sulfite oxidase of T. novellus catalyzes the oxidation of sulfite according to the following equation.